Acyl-coenzyme A carboxylases (ACCases), such as acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC), catalyze the carboxylation of acetyl- and propionyl-CoA to provide malonyl- and methylmalonyl-CoA, respectively. This carboxylation reaction is ubiquitously important in biological systems, because it commits acetyl-CoA and propionyl-CoA to the biosyntheses of fatty acids, polyketides and Kreb cycle intermediates. While there is a well-developed body of knowledge on the genetic analysis, mechanistic and biomimetic studies of ACCases, the development of ACCase-related therapeutics has been severely hampered by the lack of molecular information on how ACCases recognize their corresponding substrates or inhibitors. Our long-term goal is to generate ACCase-based therapeutics and to screen for their pharmaceutical activities. The objective of this particular application, which is the next step toward our long-term goal, is to determine the molecular basis of substrate specificity of ACC and PCC from Streptomyces coelicolor. The S. coelicolor ACCases provide extender units to the biosynthesis of polyketides, a class of natural products that include many antibiotic, anticancer and cholesterol-lowering pharmaceuticals. Mutant ACCases can potentially provide new building blocks to polyketide biosynthesis, so that new polyketides with altered extender units can be biosynthesized. These new polyketides, with the antibiotic chemical templates, will be excellent drug leads to be screened against bioterrorism targets of bacteria and viruses. The central hypothesis is that it should be possible to use mutagenesis to change the substrate specificity of ACCase for the purpose of generating new extender units for polyketide biosynthesis. We base the hypothesis on the observation that 1) ACCase subunits have distinct specificity for different substrate and inhibitors;2) our preliminary data on the structures and functions of the ACCase 2-subunits (AccB and PccB) have identified specific residues that are responsible for molecular recognition. If the hypothesis is true, mutant ACCases will produce new substituted malonyl-CoAs, which can serve as new extender units for polyketide biosynthesis. We will pursue two specific aims: AIM 1. SOLVE COCRYSTAL STRUCTURES OF ACCB AND PCCB: 1.1. Solve protein-substrate cocrystal structures. 1.2. Solve protein-regulator cocrystal structures. AIM 2. MAKE ACTIVE SITE MUTANTS OF ACCB AND PCCB: 2.1. Systematically mutate residue 422. 2.2. Mutate residues in the acyl-CoA binding pocket. 2.3. Mutate residues in the biotin binding pocket. Once we identify the residues that can be mutated to change the specificity of ACCase, it will become possible to generate new, substituted malonyl-CoAs that can serve as new extender units for polyketide biosynthesis. This innovative approach has not been undertaken before. Because of our research focus and the complementary expertise, our research environment is especially conductive to successful completion of the proposed investigations on ACCases. The research proposed in this application is significant, because its outcome allows us to dissect the molecular features that are responsible for substrate specificity of ACCases. In the long run, the result from this proposal will have a significant positive impact on the development of new antibiotics that are either ACCase inhibitors (for blocking fatty acid biosynthesis of bacteria) or have new extender units (for the biosynthesis of new polyketides). Finally, the molecular basis of substrate specificity, determined from the proposed research, will mark a breakthrough in the research of acyl-CoA carboxylase. PUBLIC HEALTH RELEVANCE: This project will result in the production of new polyketides that are synthesized with new building blocks. Because polyketides contain many antibiotic and anticancer compounds, the outcome of this project will benefit the general public health by providing new "unnatural" natural products for new drug leads.